Berglund:Native Acrylamide

For an excellent review on acrylamide and problems associated with acrylamide gels (native and denaturing) please refer to: www.bio-rad.com/LifeScience/pdf/Bulletin_1156.pdf

To make a 6% native acrylamide gel used for binding gels (Rig 4, 30-60 wells) :

26ml 5xTB
19.8ml 40% acrylamide (37.5:1) (Biorad 161-0148)
84ml water (nanopure)

Mix well

Add 1ml 10% ammonium persulfate (VWR em-2310)
Mix well

Add 150µl Temed (Biorad 161-0800).

Pour quickly.

Ammonium persulfate (APS) is a catalyst for acrylamide gel polymerization. Used in conjunction with TEMED to promote polymerization of Acrylamide/bis-Acrylamide gels. We make a 10ml solution in a 15ml falcon tube and can use it over the period of one week. This method does NOT work well if you are running in vivo splicing gels (which have their own seperate protocol).

The ratio of acrylamide to bis-acrylamide dictates the pore size, the sieving of 37.5:1 is much less than the tighter pore 19:1. This makes 37.5:1 ideal for large complexes of protein and RNA.

The buffer TB (to make a 5x solution= 216g Tris base, 110g Boric Acid fv= 5L) does not include EDTA. Do not use EDTA with a binding gel because EDTA can chelate the Mg away and smear complexes. DON'T use TB buffer in your gel and TBE buffer as your running buffer. DON'T use 0.5xTB in your gel and 1xTB in your running buffer. This spells disaster. The better the match from gel to running buffer the crisper your bands.

Other additives you can add to the gel: magnesium acetate, glycerol. Match the concentrations to that in your binding conditions (ie if you use 2mM MgCl2 in your binding, use 2mM MgOAc; 5% glycerol in your binding= 5% glycerol in your gel). This can help with well shifting and with smearing bands.

Clean your glass plates if they are dirty with a 200proof Methanol solution saturated with KOH. Wear gloves, pour solution on glass, scrape yuck off the glass with a razor (don't scratch the glass). Rinse REALLY well with water. When the glass is clean you can tell because it will be a sheet of water on the surface instead of blobs of water.

You can also silanize one side of one of the glass plates. Don't silanize both glass plates, your gel will fall out.